Plasmid autonomously replicable in coryneform bacteria

ABSTRACT

A plasmid isolatable from  Corynebacterium thermoaminogenes , which comprises a gene coding for a Rep protein having the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having homology of 90% or more to the amino acid sequence shown in SEQ ID NO: 8, and has a size of about 4.4 kb or about 6 kb, or a derivative thereof.

[0001] This application is a divisional of Ser. No. 09/636,458, filed Aug. 11, 2000, which is hereby incorporated by reference. All documents cited herein, as well as the foreign priority document, JP1999-228391, filed Aug. 12, 1999, are also hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to a novel plasmid derived from Corynebacterium thermoaminogenes. The plasmid of the present invention can be utilized for improving coryneform bacteria, which are used for producing useful substances such as L-amino acids.

[0003] Amino acids, including L-glutamic acid and L-lysine, are produced by fermentative methods using the coryneform bacteria, which generally belong to the genus Brevibacterium, Corynebacterium, or Microbacterium, or variant strains thereof (Amino Acid Fermentation, Gakkai Shuppan Center, pp. 195-215, 1986).

[0004] In the industrial fermentative production of amino acids, besides improving the yield relative to saccharides, shortening the culture time, improving the amino acid concentration, and so forth, increasing the culture temperature is an important technical factor that increases the economical efficiency. That is, the culture is usually performed at an optimum fermentation temperature, which is 31.5° C. for Corynebacterium glutamicum. After the culture is started, heat is generated during the fermentation, and hence amino acid production is markedly reduced if this heat output is not removed. Therefore, cooling equipment is required in order to maintain the optimum temperature of the culture broth. On the other hand, if the culture temperature can be elevated, it is then possible to decrease the energy required for cooling and the cooling equipment can be reduced in size.

[0005] Among coryneform bacteria, Corynebacterium thermoaminogenes has been isolated as a coryneform bacterium that can grow in higher temperatures (Japanese Patent Application Laid-open (Kokai) No. 63-240779). Whereas growth of Corynebacterium glutamicum is markedly suppressed at 40° C., Corynebacterium thermoaminogenes can grow at a temperature of about 40° C. or higher, and is therefore suitable for high temperature fermentation.

[0006] Currently, reliability of DNA recombination techniques is steadily improving in Escherichia coli and coryneform bacteria. To improve microorganisms using DNA recombinant techniques, plasmids derived from microorganisms belonging to other species, genus or broad host spectrum vectors are often used. However, plasmids native to the objective microorganism are generally used. In particular, when the optimum culture temperature for the objective microorganism to be improved is different from that of a microorganism of the same species or genus, it is preferable to use a plasmid native to the microorganism.

[0007] To date, plasmids derived from coryneform bacteria which have been obtained are pAM330 from Brevibacterium lactofermentum ATCC13869 (Japanese Patent Application Laid-open (Kokai) No. 58-67669), pBL1 from Brevibacterium lactofermentum ATCC21798 (Santamaria. R. et al., J. Gen. Microbiol., 130, pp.2237-2246, 1984), pHM1519 from Corynebacterium glutamicum ATCC13058 (Japanese Patent Application Laid-open (Kokai) No. 58-77895), pCG1 from Corynebacterium glutamicum ATCC31808 (Japanese Patent Application Laid-open (Kokai) No. 57-134500) and pGA1 from Corynebacterium glutamicum DSM58 (Japanese Patent Application Laid-open (Kokai) No. 9-2603011).

[0008] However, no plasmid native to Corynebacterium thermoaminogenes has been obtained at present.

SUMMARY OF THE INVENTION

[0009] An object of the present invention is to provide a plasmid which is useful for improving a coryneform bacterium that can grow at an elevated temperature, Corynebacterium thermoaminogenes.

[0010] The inventors of the present invention found that Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539), AJ12308 (FERM BP-1540), AJ12309 (FERM BP-1541) and AJ12310 (FERM BP-1542) each harbored a cryptic plasmid native to each strain, and successfully isolated and identified each plasmid. Thus, they accomplished the present invention.

[0011] That is, the present invention provides a plasmid isolatable from Corynebacterium thermoaminogenes, which comprises a gene (rep gene) coding for a Rep protein which has the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence which has homology of 90% or more to the foregoing amino acid sequence, and has a size of about 4.4 kb or about 6 kb, or a derivative thereof.

[0012] Examples of the aforementioned plasmids include a plasmid isolatable from Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539), AJ12308 (FERM BP-1540) or AJ12310 (FERM BP-1542), which has a size of about 4.4 kb and is depicted in the restriction map shown in FIG. 1, and a plasmid isolatable from Corynebacterium thermoaminogenes AJ12309 (FERM BP-1541), which has a size of about 6 kb and is depicted in the restriction map shown in FIG. 2.

[0013] Specific examples of the aforementioned plasmid include a plasmid which comprises a gene coding for a Rep protein having the amino acid sequence shown in SEQ ID NO: 2, 4 or 6, and a plasmid which comprises a gene coding for a Rep protein having the amino acid sequence shown in SEQ ID NO: 8.

BRIEF EXPLANATION OF THE DRAWINGS

[0014]FIG. 1 is a restriction map of the plasmids pYM1, pYM2 and pYM3 of the present invention.

[0015]FIG. 2 is a restriction map of the plasmid pYM4 of the present invention.

[0016]FIG. 3 shows construction of pYMFK.

[0017]FIG. 4 shows construction of pYMK.

[0018]FIG. 5 shows construction of pYMC.

[0019]FIG. 6 shows construction of pK1.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The plasmid of the present invention can be isolated from Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539), AJ12308 (FERM BP-1540), AJ12309 (FERM BP-1541) or AJ12310 (FERM BP-1542) according to a usual method for preparing a plasmid, such as the alkali method (Text for Bioengineering Experiments, Edited by the Society for Bioscience and Bioengineering, Japan, p. 105, Baifukan, 1992). FERM BP-1539 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (postal code 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on Mar. 13, 1987 and given an accession number of FERM P-9277, and was transferred to an international depository and deposited under the provisions of the Budapest Treaty on Oct. 27, 1987. FERM BP-1540, FERM BP-1541 and FERM BP-1542 were deposited at the aforementioned depository on Mar. 10, 1987 and given accession numbers of FERM P-9244, FERM P-9245 and FERM P-9246, and were transferred to an international depository and deposited under the provisions of the Budapest Treaty on Oct. 27, 1987.

[0021] The inventors of the present invention isolated and identified plasmids native to each of the aforementioned Corynebacterium thermoaminogenes AJ12308 (FERM BP-1540), AJ12310 (FERM BP-1542), AJ12340 (FERM BP-1539) and AJ12309 (FERM BP-1541), and designated them as pYM1, pYM2, pYM3 and pYM4, respectively. These plasmids exist as double-stranded circular DNA in a cell of Corynebacterium thermoaminogenes. The nucleotide sequence of the rep gene contained in pYM1 is shown in SEQ ID NO: 1, the nucleotide sequence of the rep gene contained in pYM2 is shown in SEQ ID NO: 3, the nucleotide sequence of the rep gene contained in pYM3 is shown in SEQ ID NO: 5, and the nucleotide sequence of the rep gene contained in pYM4 is shown in SEQ ID NO: 7. The amino acid sequences that can be encoded by the rep genes contained in these plasmids are shown in SEQ ID NOS: 2, 4, 6 and 8. pYM1, pYM2 and pYM3 each have a size of about 4.4 kb. pYM4 has a size of about 6 kb.

[0022] The numbers and sizes of fragments that can be obtained when pYM1, pYM2 and pYM3 are digested with typical restriction enzymes are shown in Table 1. The numbers and sizes of fragments that can be obtained when pYM4 is digested with typical restriction enzymes are shown in Table 2. Further, a restriction map of pYM1, pYM2 and pYM3 is shown in FIG. 1, and a restriction map of pYM4 is shown in FIG. 2. TABLE 1 Restriction Number of DNA fragment enzyme digestion site (kb) BglII 0 — BamHI 2 1.8, 2.6 BstPI 1 4.4 EcoRI 1 4.4 HincII 4 0.3, 0.5, 2.0, 1.6 HindIII 0 — KpnI 0 — NaeI 2 0.1, 4.3 NcoI 1 4.4 NheI 2 1.8, 2.6 PmaCI 1 4.4 SacI 0 — SalI 0 — SacII 3 0.1, 1.4, 2.9 SmaI 3 0.1, 1.8, 2.5 SphI 0 — Tth111I 1 4.4 XbaI 0 —

[0023] TABLE 2 Restriction Number of DNA fragment enzyme digestion site (kb) BglII 1 6.0 BamHI 2 3.8, 2.2 BstPI 2 1.2, 4.8 EcoRI 1 6.0 HincII 4 0.3, 0.4, 1.2, 1.7, 2.4 HindIII 0 — KpnI 0 — NaeI 2 0.1, 5.9 NcoI 3 0.2, 2.8, 3.0 NheI 3 0.1, 2.3, 3.6 PmaCI 0 — SacI 0 — SalI 0 — SacII 5 0.1, 0.2, 0.9, 1.8, 3.0 SmaI 2 0.1, 5.9 SphI 0 — Tth111I 0 — XbaI 0 —

[0024] Determination of the nucleotide sequence of the plasmids of the present invention revealed that pYM1, pYM2, and pYM3 each contain 4368 bp, 4369 bp and 4369 bp, respectively, have substantially the same structure, and have homology of 99.9% to one another on the nucleotide sequence level. Further, pYM4 contains 5967 bp and has extremely high homology to pYM1, pYM2 and pYM3 in the about 4.4 kb region, while pYM4 only has homology of about 81% when compared as a whole.

[0025] The plasmids contain respective rep genes which have high homology to one another. Homology was compared for the amino acid sequences of the Rep proteins encoded by the rep genes (SEQ ID NOS: 2, 4, 6 and 8) and the amino acid sequences of the Rep proteins encoded by rep genes of known plasmids derived from coryneform bacteria. Homology of 99% or more was observed among pYM1, pYM2 and pYM3, and homology of 81.91% was observed between pYM2 and pYM4. On the other hand, they showed no homology to the known plasmid pAM330 of a coryneform bacterium, and they showed homology of 80% or less to pGA1 and pCG1. The results are shown in Table 3. Thus, the plasmid of the present invention and the known plasmids of coryneform bacteria are distinguishable based on the homology of the Rep protein.

[0026] The homology is calculated according to the method described in Takashi, K. and Gotoh, O., J. Biochem., 92, 1173-1177 (1984). TABLE 3 Homology of amino acid sequences of Rep protein encoded by various plasmids PYM2 pYM4 pGA1 pCG1 PYM2 — 81.91% 68.01% 70.73% PYM4 — — 69.39% 70.23% PGA1 — — — 75.31% PCG1 — — —   —

[0027] Since the plasmid of the present invention can sufficiently replicate in cells of coryneform bacteria, including Corynebacterium thermoaminogenes, the genetic information of a foreign gene can be expressed in a host microorganism by inserting the foreign gene at any site in the plasmid, or the derivative thereof, and transforming the host microorganism with the resulting recombinant plasmid.

[0028] Examples of coryneform bacteria are listed below.

[0029]Corynebacterium acetoacidophilum

[0030]Corynebacterium acetoglutamicum

[0031]Corynebacterium callunae

[0032]Corynebacterium glutamicum

[0033]Corynebacterium thermoaminogenes

[0034]Corynebacterium lilium (Corynebacterium glutamicum)

[0035]Corynebacterium melassecola

[0036]Brevibacterium divaricatum (Corynebacterium glutamicum)

[0037]Brevibacterium lactofermentum (Corynebacterium glutamicum)

[0038]Brevibacterium saccharolyticum

[0039]Brevibacterium immariophilum

[0040]Brevibacterium roseum

[0041]Brevibacterium flavum (Corynebacterium glutamicum)

[0042]Brevibacterium thiogenitalis

[0043] A “derivative” of the plasmid of the present invention means a plasmid composed of a part of the plasmid of the present invention, or the plasmid of present invention and another DNA sequence. The “part of a plasmid” means a part containing a region essential for autonomous replication of the plasmid. The plasmid of the present invention can replicate in a host microorganism even if a region other than the region essential for the autonomous replication of the plasmid (replication control region), that is, the region other than the region containing the replication origin and genes necessary for the replication, is deleted. In addition, a plasmid having such a deletion will have a smaller size. Therefore, a plasmid having such a deletion is preferred for use as a vector. Furthermore, if a marker gene, such as a drug resistance gene, is inserted into the plasmid of the present invention or a part thereof, it becomes easy to detect transformants thanks to the phenotype of the marker gene in the transformants. Examples of such a marker gene that can be used in the host include chloramphenicol resistance gene, kanamycin resistance gene, streptomycin resistance gene, tetracycline resistence gene, trimethoprim resistance gene, erythromycin resistance gene, and so forth.

[0044] Furthermore, if the plasmid of the present invention is made as a shuttle vector, which is autonomously replicable in coryneform bacteria and other bacteria such as Escherichia coli, by ligating the plasmid of the present invention or a part thereof with a plasmid autonomously replicable in the other bacteria such as Escherichia coli or a part thereof containing a replication control region thereof, manipulations can be performed using Escherichia coli, such as preparation of plasmid and preparation of recombinant plasmid containing a target gene. Examples of a plasmid autonomously replicable in Escherichia coli include, for example, pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF110, pMW119, pMW118, pMW219, pMW218, and so forth.

[0045] Although pYM1, pYM2, pYM3 and pYM4 are characterized by the restriction maps shown in FIGS. 1 and 2, it is not necessarily required that the plasmid of present invention have these restriction maps, and any restriction site may be deleted as long as such deletion does not affect the autonomous replication ability. Furthermore, the plasmid of the present invention may contain a restriction site that is not contained in pYM1, pYM2, pYM3 and pYM4.

[0046] The derivative of the plasmid as described above can be constructed in the same manner as the conventionally known construction of cloning vectors, expression vectors and so forth. In order to construct the derivative, it is preferable to determine the nucleotide sequences of pYM1, pYM2, pYM3 and pYM4. The nucleotide sequences can be determined by known methods, such as the dideoxy method.

[0047] In order to insert a foreign gene into the plasmid or the derivative thereof of the present invention, it is convenient to insert it into a restriction site of the plasmid or the derivative thereof. A restriction site which is present as a single digestion site is preferred. In order to insert a foreign gene, the plasmid and the source of the foreign gene, such as genomic DNA, can be partially or fully digested with one or more restriction enzymes that provide the same cohesive ends, e.g., the same restriction enzyme, and they can be ligated under suitable conditions. They may also be blunt-end ligated.

[0048] For the preparation of plasmid DNA, digestion and ligation of DNA, transformation and so forth, methods well-known to those skilled in the art may be employed. Such methods are described in Sambrook, J., Fritsch, E. F., and Maniatis, T., “Molecular Cloning: A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989), and so forth.

[0049] According to the present invention, a novel plasmid derived from Corynebacterium thermoaminogenes is provided as described above.

EXAMPLES

[0050] Hereinafter, the present invention will be explained in more detail with reference to the following examples.

Example 1

[0051] Isolation and Characterization of Plasmids from Corynebacterium Thermoaminogenes (FERM BP-1539, FERM BP-1540, FERM BP-1541, FERM BP-1542)

[0052]Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539), AJ12308 (FERM BP-1540), AJ12309 (FERM BP-1541) and AJ12310 (FERM BP-1542) were cultured for 12 hours in CM2B liquid medium (Bacto-trypton (Difco): 1%, Bacto-yeast-extract (Difco): 1%, NaCl: 0.5%, biotin: 10 μg/L), and plasmid DNA fractions were obtained by the alkali method (Text for Bioengineering Experiments, Edited by the Society for Bioscience and Bioengineering, Japan, p.105, Baifukan, 1992). When these fractions were analyzed by agarose gel electrophoresis (Sambrook, J., Fritsch, E. F., and Maniatis, T., “Molecular Cloning: A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989)), DNA bands were detected for all of the fractions, and hence it was demonstrated that the aforementioned strains harbored plasmids. The plasmids prepared from FERM BP-1540, FERM BP-1542 and FERM BP-1539 were designated as pYM1, pYM2 and pYM3, respectively. The plasmid prepared from FERM BP-1541 was designated as pYM4. The plasmids pYM1, pYM2 and pYM3 each had a length of about 4.4 kb, and the plasmid pYM4 had a length of about 6.0 kb.

[0053] The plasmids pYM1, pYM2, pYM3 and pYM4 were digested with restriction enzymes BglII, BamHI, BstPI, EcoRI, HincII, HindIII, KpnI, NaeI, NcoI, NheI, PmaCI, SacI, SacII, SalI, SmaI, SphI, Tth111I and XbaI (produced by Takara Co.), and the lengths of the produced DNA fragments were measured by agarose gel electrophoresis. The electrophoresis was performed at 100 V/cm and a constant voltage for several hours by using a 0.8% agarose gel. λ phage DNA (Takara Shuzo) digested with a restriction enzyme HindIII was used as molecular weight markers. The results obtained for pYM1, pYM2 and pYM3 are shown in Table 1. The results obtained for pYM4 are shown in Table 2. The restriction map of pYM1, pYM2 and pYM3 is shown in FIG. 1, and the restriction map of pYM4 is shown in FIG. 2, which were prepared based on the above results.

[0054] The results of nucleotide sequencing of pYM1, pYM2, pYM3 and pYM4 by the dideoxy method are shown in SEQ ID NOS: 1, 3, 5 and 7 respectively.

Example 2

[0055] Construction of the Shuttle Vector pYMFK Containing the Km Resistance Gene Derived from Streptococcus Faecalis

[0056] Regions necessary for efficient replication of pYM2 in coryneform bacteria include an AT-rich region upstream from rep and a region which affects copy number downstream from rep, besides the region coding for rep.

[0057] Therefore, in order to obtain a shuttle vector that can replicate in coryneform bacteria and E. coli without impairing the replication ability of pYM2, a region enabling autonomous replication in E. coli and a selection marker were inserted into sites in the vicinity of the BstPI site of pYM2.

[0058] First, a vector having a drug resistance gene of S. faecalis was constructed. The kanamycin resistance gene of S. faecalis was amplified by PCR from a known plasmid containing that gene. The nucleotide sequence of the kanamycin resistance gene of S. faecalis has already been elucidated (Trieu-Cuot, P. and Courvalin, P., Gene, 23 (3), pp.331-341 (1983)). Based on this sequence, primers having the nucleotide sequences shown as SEQ ID NOS: 16 and 17 were synthesized, and PCR was performed using pDG783 (Anne-Marie Guerout-Fleury et al., Gene, 167, pp.335-337 (1995)) as a template to amplify a DNA fragment containing the kanamycin resistance gene and its promoter.

[0059] The above DNA fragment was purified by using SUPREC02 produced by Takara Shuzo Co., Ltd., completely digested with restriction enzymes HindIII and HincII, and blunt-ended. The blunt-ending was performed by Blunting Kit produced by Takara Shuzo Co., Ltd. This DNA fragment and an amplification product obtained by PCR with primers having the nucleotide sequences shown as SEQ ID NOS: 18 and 19, and pHSG399 (see S. Takeshita et al., Gene, 61, pp.63-74 (1987)) as a template, and purification and blunt-ending were mixed and ligated. The ligation reaction was performed by using DNA Ligation Kit ver.2 produced by Takara Shuzo Co., Ltd. Competent cells of Escherichia coli JM109 (produced by Takara Shuzo Co., Ltd.) were transformed with the ligated DNA, and cultured overnight in L medium (10 g/L of Bacto trypton, 5 g/L of Bacto yeast extract, 5 g/L of NaCl, and 15 g/L of agar, pH 7.2) containing 10 μg/ml of IPTG (isopropyl-β-D-thiogalactopyranoside), 40 μg/ml of X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) and 25 μg/ml of kanamycin. Then, the formed blue colonies were subjected to single colony isolation to obtain transformants.

[0060] Plasmids were prepared from the transformants using the alkaline method (Text for Bioengineering Experiments, Edited by the Society for Bioscience and Bioengineering, Japan, p.105, Baifukan, 1992), and restriction maps were prepared. A plasmid having a restriction map equivalent to that shown at a lower position in FIG. 6 was designated as pK1. This plasmid is stably harbored in Escherichia coli, and imparts kanamycin resistance to a host. Moreover, since it contains the lacZ′ gene, it is suitable for use as a cloning vector.

[0061] Then, a region containing the replication origin was amplified by Pyrobest-Taq (Takara Shuzo Co., Ltd.) using pYM2 extracted from C. thermoaminogenes AJ12310 (FERM BP-1542) as a template (The entire nucleotide sequence of pYM2 is shown in SEQ ID NO: 9.) and the following primers were prepared based on a sequence in pYM2 near the BstPI site:

[0062] S1: 5′-AAC CAG GGG GAG GGC GCG AGG C-3′ (SEQ ID NO: 10)

[0063] S3: 5′-TCT CGTAGG CTG CAT CCGAGG CGG GG-3′ (SEQ ID NO: 11)

[0064] The reaction conditions were 94° C. for 5 minutes, followed by a cycle of 98° C. for 20 seconds, and 68° C. for 4 minutes, which was repeated for 30 cycles, and 72° C. for 4 minutes. After the reaction, the mixture was stored at 4° C.

[0065] The resulting amplified fragment was purified using MicroSpin TM S-400 HR columns produced by Amersham Pharmacia Biotech Co., blunt-ended using DNA Blunting Kit produced by Takara Shuzo Co., Ltd., and then ligated to pK1, which had been treated with HincII using DNA Ligation Kit. ver. 2 produced by Takara Shuzo Co., Ltd. Competent cells of Escherichia coli JM109 (produced by Takara Shuzo) were transformed with the ligated DNA to obtain transformant strains.

[0066] Plasmids were prepared from the transformant strains using the alkali method (Text for Bioengineering Experiments, Edited by the Society for Bioscience and Bioengineering, Japan, p.105, Baifukan, 1992) and restriction maps of the plasmids were prepared. A restriction map equivalent to that shown at a lower position in FIG. 3 was designated as pYMFK. pYMFK had a size of about 7.0 kb, and was able to autonomously replicate in E. coli and coryneform bacteria and impart Km resistance to a host.

Example 3

[0067] Construction of pYMK Containing Km Resistance Gene Derived from Tn903

[0068] A region containing the replication origin was amplified in the same manner as in Example 2 by using pYM2 extracted from C. thermoaminogenes AJ12310 (FERM BP-1542) as a template and the following primers: S1XbaI: 5′-GCT CTA GAG CAA CCA GGG GGA GGG CGC GAG GC-3′ (SEQ ID NO: 12) S3XbaI: 5′-GCT CTA GAG CTC TCG TAG GCT GCA TCG GAG GCG GGG-3′ (SEQ ID NO: 13)

[0069] The obtained amplified fragment was purified by using MicroSpin TM S-400 HR columns produced by Amersham Pharmacia Biotech Co., digested with a restriction enzyme XbaI produced by Takara Shuzo Co., Ltd., and then ligated to a fragment obtained by fully digesting pHSG299 (Takara Shuzo Co., Ltd.) with XbaI by using DNA Ligation Kit. ver. 2 produced by Takara Shuzo Co., Ltd. Competent cells of Escherichia coli JM109 (produced by Takara Shuzo) were transformed with the ligated DNA to obtain transformant strains.

[0070] Plasmids were prepared from the transformant strains using the alkali method (Text for Bioengineering Experiments, Edited by the Society for Bioscience and Bioengineering, Japan, p.105, Baifikan, 1992) and restriction maps of the plasmids were prepared. A restriction map equivalent to that shown at a lower position in FIG. 4 was designated as pYMK. pYMK had a size of about 7.0 kb, and was able to autonomously replicate in E. coli and coryneform bacteria and impart Km resistance to a host.

Example 4

[0071] Construction of Shuttle Vector pYMC Containing Cm Resistance Gene Derived from Tn9

[0072] A region containing the replication origin was amplified in the same manner as in Example 2 by using pYM2 extracted from C. thermoaminogenes AJ12310 (FERM BP-1542) as a template and the following primers: S1XbaI: 5′-GCT CTA GAG CAA CCA GGG GGA GGG CGC GAG GC-3′ (SEQ ID NO: 14) S3XbaI: 5′-GCT CTA GAG CTC TCG TAG GCT GCA TCG GAG GCG GGG-3′ (SEQ ID NO: 15)

[0073] The above DNA was purified by using MicroSpin TM S-400 HR columns produced by Amersham Pharmacia Biotech Co., digested with a restriction enzyme XbaI produced by Takara Shuzo Co., Ltd., and then ligated to a fragment obtained by treating pHSG399 (Takara Shuzo Co., Ltd.) with XbaI using DNA Ligation Kit. ver. 2 produced by Takara Shuzo Co. Ltd. Competent cells of Escherichia coli JM109 (produced by Takara Shuzo) were transformed with the ligated DNA to obtain transformant strains.

[0074] Plasmids were prepared from the transformant strains using the alkali method (Text for Bioengineering Experiments, Edited by the Society for Bioscience and Bioengineering, Japan, p. 105, Baifukan, 1992) and restriction maps of the plasmids were prepared. One showing a restriction map equivalent to that shown at a lower position in FIG. 5 was designated as pYMC. pYMC had a size of about 6.6 kb, and was able to autonomously replicate in E. coli and coryneform bacteria and impart Cm resistance to a host.

1 20 1 1479 DNA Corynebacterium thermoaminogenes CDS (1)..(1476) 1 atg act cta gcg gat tcg cca gga aca tac aca gca gat gcg tgg aat 48 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 tac tcc act gat ctg ttc gac acc cac cct gag ctg gct tta cgc tcc 96 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 cgg ggt tgg aat cac cag gac gcc gcc gag ttc ctg gcc cac ctg gat 144 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 cgc agc atg ttt cac ggg tgc ccc acc cgg gat ttc tcc gcg gcc tgg 192 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp 50 55 60 gtc aaa gac ccg gaa acc gga gaa acc cgc ccc aag ctg cac aga gtt 240 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 ggc acc cgc tca ctt tcc cgg tgc cag tac gtt gcc ctg acc cac ccg 288 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 cag cgc tcc gcg gtg ctg gtc tta gac atc gac atc ccc agc cac cag 336 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 gcc ggc ggg aac atc gag cac ctt cac ccg cag gtg tac gcc acc ttg 384 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 gag cgt tgg gca cgg gtg gag aaa gcg ccg gcc tgg atc ggg gtg aac 432 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 ccg ttg tcg gga aag tgc cag ctc atc tgg tgc att gac ccg gtg ttc 480 Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe 145 150 155 160 gcc gcc gag ggc acc acc agc tcg aac acc cgc ctg cta gcg gcc acc 528 Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 acc gag gaa atg acc cgt gtg ttc ggc gct gac cag gca ttt tcc cac 576 Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 cgg ctg agc cgg tgg ccg ctg cat gtt tct gat gat ccg acc gcg tac 624 Arg Leu Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr 195 200 205 tcc tgg cac tgc cag cac aac cga gtc gat att ctt gat gag ctg atg 672 Ser Trp His Cys Gln His Asn Arg Val Asp Ile Leu Asp Glu Leu Met 210 215 220 gag gta gcc cgc acg atg acc gga tca aaa aag ccc aga gag cac gct 720 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Glu His Ala 225 230 235 240 cac cag gag ttt tcc agc ggt cgg gca cgg atc gaa gcc gcg cgg aaa 768 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 gcc acc gca gag gcc aaa gcg ctt gcc gcc ttg gac gcc acg ctg cct 816 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 acg gcg ctg gag gca tca ggc gat ctc att gac ggg gtg cgg gtg ttg 864 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 285 tgg gca gca gag ggg cgt gca gcc cgt gat gag aca gcg ttt cgc cat 912 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 gcg ttg acc gtg ggt tat cag ctt aaa gcc gca ggt gaa cgc ctg aaa 960 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 gat gcc aag atc att gat gcg tat gag cgt gcc tac aac gtc gcc cag 1008 Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln 325 330 335 gcg gtg gga gct gat ggg cgt gaa ccg gat ctg cct gcc atg cgt gat 1056 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 cgt cag acg atg gcc cgc cgt gtg cgc gcc tac gtc gcc aaa ggc cag 1104 Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 ccc acg gtc agc gcc agg agc aca cag acc cag agc agt cgg ggc cgg 1152 Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 aaa gcc ctg gcc acc atg ggc cgc aga ggc ggg caa aaa gcc gct gaa 1200 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu 385 390 395 400 cgc tgg aaa acc gat cct aac ggc aaa tac gcc caa gaa aac cgc caa 1248 Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln 405 410 415 cga ctc gaa gct gca aac aag cga cgt caa gtc agc tgg aac aaa tac 1296 Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr 420 425 430 gcg agc acg aat tct ggc tac ggt ttc cga cac gta tgg gcc agc ttg 1344 Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu 435 440 445 gaa aaa tgc cta cgc gac gag caa atc atg gaa gaa aca ggg ctt tca 1392 Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser 450 455 460 gaa aaa tgc cta cgc gac gag caa atc atg gaa gaa aca ggg ctt tca 1440 Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser 465 470 475 480 tgc caa atc ctt agg ggg gct cac gcc gta gac aga taa 1479 Cys Gln Ile Leu Arg Gly Ala His Ala Val Asp Arg 485 490 2 492 PRT Corynebacterium thermoaminogenes 2 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp 50 55 60 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe 145 150 155 160 Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 Arg Leu Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr 195 200 205 Ser Trp His Cys Gln His Asn Arg Val Asp Ile Leu Asp Glu Leu Met 210 215 220 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Glu His Ala 225 230 235 240 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 285 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln 325 330 335 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu 385 390 395 400 Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln 405 410 415 Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr 420 425 430 Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu 435 440 445 Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser 450 455 460 Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser 465 470 475 480 Cys Gln Ile Leu Arg Gly Ala His Ala Val Asp Arg 485 490 3 1479 DNA Corynebacterium thermoaminogenes CDS (1)..(1476) 3 atg act cta gcg gat tcg cca gga aca tac aca gca gat gcg tgg aat 48 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 tac tcc act gat ctg ttc gac acc cac cct gag ctg gct tta cgc tcc 96 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 cgg ggt tgg aat cac cag gac gcc gca gag ttc ctg gcc cac ctg gat 144 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 cgc agc atg ttt cac ggg tgc ccc acc cgg gat ttc tcc gcg gcc tgg 192 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp 50 55 60 gtc aaa gac ccg gaa acc gga gaa acc cgc ccc aag ctg cac aga gtt 240 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 ggc acc cgc tca ctt tcc cgg tgc cag tac gtt gcc ctg acc cac ccg 288 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 cag cgc tcc gcg gtg ctg gtc tta gac atc gac atc ccc agc cac cag 336 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 gcc ggc ggg aac atc gag cac ctt cac ccg cag gtg tac gcc acc ttg 384 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 gag cgt tgg gca cgg gtg gag aaa gcg ccg gcc tgg atc ggg gtg aac 432 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 ccg ttg tcg gga aag tgc cag ctc atc tgg tgc att gac ccg gtg ttc 480 Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe 145 150 155 160 gcc gcc gag ggc acc acc agc tcg aac acc cgc ctg cta gcg gcc acc 528 Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 acc gag gaa atg acc cgt gtg ttc ggc gct gac cag gca ttt tcc cac 576 Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 cgg ctg agc cgg tgg ccg ctg cat gtt ttt gat gat ccg acc gcg tac 624 Arg Leu Ser Arg Trp Pro Leu His Val Phe Asp Asp Pro Thr Ala Tyr 195 200 205 tcc tgg cac tgc cag cac aac cga gtc gat att ctt gat gag ctg atg 672 Ser Trp His Cys Gln His Asn Arg Val Asp Ile Leu Asp Glu Leu Met 210 215 220 gag gta gcc cgc acg atg acc gga tca aaa aag ccg aga aag cac gct 720 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Lys His Ala 225 230 235 240 cac cag gag ttt tcc agc ggt cgg gca cgg atc gaa gcc gcg cgg aaa 768 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 gcc acc gca gag gcc aaa gcg ctt gcc gcc ttg gac gcc acg ctg cct 816 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 acg gcg ctg gag gca tca ggc gat ctc att gac ggg gtg cgg gtg ttg 864 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 285 tgg gca gca gag ggg cgt gca gcc cgt gat gag aca gcg ttt cgc cat 912 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 gcg ttg acc gtg ggt tat cag ctt aaa gcc gca ggt gaa cgc ctg aaa 960 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 gat gcc aag atc att gat gcg tat gag cgt gcc tac aac gtc gcc cag 1008 Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln 325 330 335 gcg gtg gga gct gat ggg cgt gaa ccg gat ctg cct gcc atg cgt gat 1056 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 cgt cag acg atg gcc cgc cgt gtg cgc gcc tac gtc gcc aaa ggc cag 1104 Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 ccc acg gtc agc gcc agg agc aca cag acc cag agc agt cgg ggc cgg 1152 Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 aaa gcc ctg gcc acc atg ggc cgc aga ggc ggg caa aaa gcc gct gaa 1200 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu 385 390 395 400 cgc tgg aaa acc gat cct aac ggc aaa tac gcc caa gaa aac cgc caa 1248 Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln 405 410 415 cga ctc gaa gct gca aac aag cga cgt caa gtc agc tgg aac aaa tac 1296 Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr 420 425 430 gcg agc acg aat tct ggc tac ggt ttc cga cac gta tgg gcc agc ttg 1344 Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu 435 440 445 gaa aaa tgc cta cgc gac gag caa atc atg gaa gaa aca ggg ctt tca 1392 Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser 450 455 460 cga gct acc gtg acg cgc cat tgg gtg cac tgc gag agg ctg gcc tgc 1440 Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys 465 470 475 480 tgc caa atc ctt agg ggg gct cac gcc gta cac aga taa 1479 Cys Gln Ile Leu Arg Gly Ala His Ala Val His Arg 485 490 4 492 PRT Corynebacterium thermoaminogenes 4 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp 50 55 60 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe 145 150 155 160 Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 Arg Leu Ser Arg Trp Pro Leu His Val Phe Asp Asp Pro Thr Ala Tyr 195 200 205 Ser Trp His Cys Gln His Asn Arg Val Asp Ile Leu Asp Glu Leu Met 210 215 220 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Lys His Ala 225 230 235 240 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 285 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln 325 330 335 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu 385 390 395 400 Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln 405 410 415 Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr 420 425 430 Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu 435 440 445 Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser 450 455 460 Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys 465 470 475 480 Cys Gln Ile Leu Arg Gly Ala His Ala Val His Arg 485 490 5 1479 DNA Corynebacterium thermoaminogenes CDS (1)..(1476) 5 atg act cta gcg gat tcg cca gga aca tac aca gca gat gcg tgg aat 48 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 tac tcc act gat ctg ttc gac acc cac cct gag ctg gct tta cgc tcc 96 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 cgg ggt tgg aat cac cag gac gcc gcc gag ttc ctg gcc cac ctg gat 144 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 cgc agc atg ttt cac ggg tgc ccc acc cgg gat ttc tcc gcg gcc tgg 192 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp 50 55 60 gtc aaa gac ccg gaa acc gga gaa acc cgc ccc aag ctg cac aga gtt 240 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 ggc acc cgc tca ctt tcc cgg tgc cag tac gtt gcc ctg acc cac ccg 288 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 cag cgc tcc gcg gtg ctg gtc tta gac atc gac atc ccc agc cac cag 336 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 gcc ggc ggg aac atc gag cac ctt cac ccg cag gta tac gcc acc ttg 384 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 gag cgt tgg gca cgg gtg gag aaa gcg ccg gcc tgg atc ggg gtg aac 432 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 ccg ttg tcg gga aag tgc cag ctc atc tgg tgc att gac ccg gtg ttc 480 Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe 145 150 155 160 gcc gcc gag ggc acc acc agc tcg aac acc cgc ctg cta gcg gcc acc 528 Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 acc gag gaa atg acc cgt gtg ttc ggc gct gac cag gca ttt tcc cac 576 Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 cgg ctg agc cgg tgg ccg ctg cat gtt tct gat gat ccg acc gcg tac 624 Arg Leu Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr 195 200 205 tcc tgg cac tgc cag cac aac cga gtc gat acg ctt gat gag ctg atg 672 Ser Trp His Cys Gln His Asn Arg Val Asp Thr Leu Asp Glu Leu Met 210 215 220 gag gta gcc cgc acg atg acc gga tca aaa aag ccg aga aag cac gct 720 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Lys His Ala 225 230 235 240 cac cag gag ttt tcc agc ggt cgg gca cgg atc gaa gcc gcg cgg aaa 768 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 gcc acc gca gag gcc aaa gcg ctt gcc gcc ttg gac gcc acg ctg cct 816 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 acg gcg ctg gag gca tca ggc gat ctc att gac ggg gtg cgg gtg ttg 864 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 285 tgg gca gca gag ggg cgt gca gcc cgt gat gag aca gcg ttt cgc cat 912 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 gcg ttg acc gtg ggt tat cag ctt aaa gcc gca ggt gaa cgc ctg aaa 960 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 gat gcc aag atc att gat gcg tat gag cgt gcc tac aac gtc gcc cag 1008 Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln 325 330 335 gcg gtg gga gct gat ggg cgt gaa ccg gat ctg cct gcc atg cgt gat 1056 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 cgt cag acg atg gcc cgc cgt gtg cgc gcc tac gtc gcc aaa ggc cag 1104 Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 ccc acg gtc agc gcc agg agc aca cag acc cag agc agt cgg ggc cgg 1152 Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 aaa gcc ctg gcc acc atg ggc cgc aga ggc ggg caa aaa gcc gct gaa 1200 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu 385 390 395 400 cgc tgg aaa acc gat cct aac ggc aaa tac gcc caa gaa aac cgc caa 1248 Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln 405 410 415 cga ctc gaa gct gca aac aag cga cgt caa gtc agc tgg aac aaa tac 1296 Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr 420 425 430 gcg agc acg aat tct ggc tac ggt ttc cga cac gta tgg gcc agc ttg 1344 Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu 435 440 445 gaa aaa tgc cta cgc gac gag caa atc atg gaa gaa aca ggg ctt tca 1392 Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser 450 455 460 cga gct acc gtg acg cgc cat tgg gtg cac tgc gag agg ctg gcc tgc 1440 Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys 465 470 475 480 tgc caa atc ctt agg ggg gct cac gcc gta cac aga taa 1479 Cys Gln Ile Leu Arg Gly Ala His Ala Val His Arg 485 490 6 492 PRT Corynebacterium thermoaminogenes 6 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp 50 55 60 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe 145 150 155 160 Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 Arg Leu Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr 195 200 205 Ser Trp His Cys Gln His Asn Arg Val Asp Thr Leu Asp Glu Leu Met 210 215 220 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Lys His Ala 225 230 235 240 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 285 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln 325 330 335 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu 385 390 395 400 Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln 405 410 415 Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr 420 425 430 Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu 435 440 445 Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser 450 455 460 Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys 465 470 475 480 Cys Gln Ile Leu Arg Gly Ala His Ala Val His Arg 485 490 7 1377 DNA Corynebacterium thermoaminogenes CDS (1)..(1374) 7 atg act cta gcg gat tcg cca gga aca tac aca gca gat gcg tgg aat 48 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 tac tcc aca gat ctg ttc gac acc cac cct gag ctg gct tta cgc tcc 96 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 cgg ggt tgg aat cac cag gac gcc gcc gag ttc ctg gcc cac ctg gat 144 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 cgc agc atg ttt cac ggg tgc ccc acc cgg gat ttc tcc gcg gcc tgg 192 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp 50 55 60 gtc aaa gac ccg gag acc gga gaa acc cgc cct aag ctg cac aga gtc 240 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 ggc acc cgg tcg ctt tcc cga tgc cag tac gtc gcg ctg acc cac ccg 288 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 cag cgc tcc gcg gtg ctg gtc tta gac atc gac atc ccc agc cac cag 336 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 gcc ggc ggg aac atc gag cac ctt cac ccg cag gtc tac gcc acc ttg 384 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 gag cgc tgg gca cgg gtg gag aaa gcg ccg gcc tgg atc ggg gtg aac 432 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 ccg ttg tca gga aag tgc cag ctc atc tgg tgc att gac ccg gtg ttc 480 Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe 145 150 155 160 gcc gcc gag ggc acc acc agc ccg aac acc cgc ctg cta gcg gcc acc 528 Ala Ala Glu Gly Thr Thr Ser Pro Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 acc gag gaa atg acc cgt atg ttc ggc gct gac cag gca ttt tcc cac 576 Thr Glu Glu Met Thr Arg Met Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 cgg ctg agc cgg tgg ccg ctg cat gta tct gat gat ccg acc gcg tac 624 Arg Leu Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr 195 200 205 tcc tgg cac tgc cag cac aac cga gtc gat acg ctt gct gag ctg atg 672 Ser Trp His Cys Gln His Asn Arg Val Asp Thr Leu Ala Glu Leu Met 210 215 220 gag gta gcc cgc acg atg acc gga tca aaa aag cca gat agc act gct 720 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Asp Ser Thr Ala 225 230 235 240 cac cag gag ttt tcc agc ggt cgg gca cgg atc gaa gcc gcg agg aaa 768 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 gcc acc gca gaa gcc aaa gcg ctt gct gcc tta gac gcc acg ctg cct 816 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 acg gcg ctg gag gca tca ggc gat ctc att gac ggg gtg cgg gtg ctg 864 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 285 tgg gca gca gag ggg cgt gca gcc cgt gat gag acg gcg ttt cgc cat 912 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 gcg ttg acc gtg ggg tat cag ctt aaa gcc gca ggt gaa cgc ctg aaa 960 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 gac acc aag atc att gat gcg tat gag cgt gcc tac aac gtc gcc cag 1008 Asp Thr Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln 325 330 335 gcg gtg ggg gct gat ggg cgt gag ccg gat ctg cct gcc atg cgt gat 1056 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 cgt cag acg ttg gcc cgt cgt gtg cgc gcc tac gtc gct aaa ggc cag 1104 Arg Gln Thr Leu Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 ccc acg gtg agc gcc agg agc aca cag acc cag agc agc cgg ggc agg 1152 Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 aaa gcc ctg gcc acc atg gga cgc aga ggc gca gcc acc tcg aat gca 1200 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Ala Ala Thr Ser Asn Ala 385 390 395 400 cgc agg tgg gca gac cca gaa agc gat tac gcc cgc caa act cgg gag 1248 Arg Arg Trp Ala Asp Pro Glu Ser Asp Tyr Ala Arg Gln Thr Arg Glu 405 410 415 cgt tta gcc cga gca atg agc ttc gta cat tca gca cag acg aga aca 1296 Arg Leu Ala Arg Ala Met Ser Phe Val His Ser Ala Gln Thr Arg Thr 420 425 430 agg gcc gga tcc tgg cct acg ttt ccg agt gca agc gcc acg gtt acg 1344 Arg Ala Gly Ser Trp Pro Thr Phe Pro Ser Ala Ser Ala Thr Val Thr 435 440 445 acc cca cga gca aag aag tcg caa cgg agc tag 1377 Thr Pro Arg Ala Lys Lys Ser Gln Arg Ser 450 455 8 458 PRT Corynebacterium thermoaminogenes 8 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp 50 55 60 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe 145 150 155 160 Ala Ala Glu Gly Thr Thr Ser Pro Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 Thr Glu Glu Met Thr Arg Met Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 Arg Leu Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr 195 200 205 Ser Trp His Cys Gln His Asn Arg Val Asp Thr Leu Ala Glu Leu Met 210 215 220 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Asp Ser Thr Ala 225 230 235 240 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 285 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 Asp Thr Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln 325 330 335 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 Arg Gln Thr Leu Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Ala Ala Thr Ser Asn Ala 385 390 395 400 Arg Arg Trp Ala Asp Pro Glu Ser Asp Tyr Ala Arg Gln Thr Arg Glu 405 410 415 Arg Leu Ala Arg Ala Met Ser Phe Val His Ser Ala Gln Thr Arg Thr 420 425 430 Arg Ala Gly Ser Trp Pro Thr Phe Pro Ser Ala Ser Ala Thr Val Thr 435 440 445 Thr Pro Arg Ala Lys Lys Ser Gln Arg Ser 450 455 9 4369 DNA Corynebacterium thermoaminogenes CDS (1)..(1476) 9 atg act cta gcg gat tcg cca gga aca tac aca gca gat gcg tgg aat 48 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 tac tcc act gat ctg ttc gac acc cac cct gag ctg gct tta cgc tcc 96 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 cgg ggt tgg aat cac cag gac gcc gca gag ttc ctg gcc cac ctg gat 144 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 cgc agc atg ttt cac ggg tgc ccc acc cgg gat ttc tcc gcg gcc tgg 192 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp 50 55 60 gtc aaa gac ccg gaa acc gga gaa acc cgc ccc aag ctg cac aga gtt 240 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 ggc acc cgc tca ctt tcc cgg tgc cag tac gtt gcc ctg acc cac ccg 288 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 cag cgc tcc gcg gtg ctg gtc tta gac atc gac atc ccc agc cac cag 336 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 gcc ggc ggg aac atc gag cac ctt cac ccg cag gtg tac gcc acc ttg 384 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 gag cgt tgg gca cgg gtg gag aaa gcg ccg gcc tgg atc ggg gtg aac 432 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 ccg ttg tcg gga aag tgc cag ctc atc tgg tgc att gac ccg gtg ttc 480 Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe 145 150 155 160 gcc gcc gag ggc acc acc agc tcg aac acc cgc ctg cta gcg gcc acc 528 Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 acc gag gaa atg acc cgt gtg ttc ggc gct gac cag gca ttt tcc cac 576 Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 cgg ctg agc cgg tgg ccg ctg cat gtt ttt gat gat ccg acc gcg tac 624 Arg Leu Ser Arg Trp Pro Leu His Val Phe Asp Asp Pro Thr Ala Tyr 195 200 205 tcc tgg cac tgc cag cac aac cga gtc gat att ctt gat gag ctg atg 672 Ser Trp His Cys Gln His Asn Arg Val Asp Ile Leu Asp Glu Leu Met 210 215 220 gag gta gcc cgc acg atg acc gga tca aaa aag ccg aga aag cac gct 720 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Lys His Ala 225 230 235 240 cac cag gag ttt tcc agc ggt cgg gca cgg atc gaa gcc gcg cgg aaa 768 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 gcc acc gca gag gcc aaa gcg ctt gcc gcc ttg gac gcc acg ctg cct 816 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 acg gcg ctg gag gca tca ggc gat ctc att gac ggg gtg cgg gtg ttg 864 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 285 tgg gca gca gag ggg cgt gca gcc cgt gat gag aca gcg ttt cgc cat 912 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 gcg ttg acc gtg ggt tat cag ctt aaa gcc gca ggt gaa cgc ctg aaa 960 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 gat gcc aag atc att gat gcg tat gag cgt gcc tac aac gtc gcc cag 1008 Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln 325 330 335 gcg gtg gga gct gat ggg cgt gaa ccg gat ctg cct gcc atg cgt gat 1056 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 cgt cag acg atg gcc cgc cgt gtg cgc gcc tac gtc gcc aaa ggc cag 1104 Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 ccc acg gtc agc gcc agg agc aca cag acc cag agc agt cgg ggc cgg 1152 Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 aaa gcc ctg gcc acc atg ggc cgc aga ggc ggg caa aaa gcc gct gaa 1200 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu 385 390 395 400 cgc tgg aaa acc gat cct aac ggc aaa tac gcc caa gaa aac cgc caa 1248 Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln 405 410 415 cga ctc gaa gct gca aac aag cga cgt caa gtc agc tgg aac aaa tac 1296 Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr 420 425 430 gcg agc acg aat tct ggc tac ggt ttc cga cac gta tgg gcc agc ttg 1344 Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu 435 440 445 gaa aaa tgc cta cgc gac gag caa atc atg gaa gaa aca ggg ctt tca 1392 Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser 450 455 460 cga gct acc gtg acg cgc cat tgg gtg cac tgc gag agg ctg gcc tgc 1440 Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys 465 470 475 480 tgc caa atc ctt agg ggg gct cac gcc gta cac aga taacggttcc 1486 Cys Gln Ile Leu Arg Gly Ala His Ala Val His Arg 485 490 caccccgtag gggtagcgct tggtccctga agctccggct cccatccctc ctcagcactc 1546 cctccccgag gggggggctc acgccgtaga cagataacgg ttcccacccc gtaggggtag 1606 cgcttggtcc ctgaagctct cacttctggc tcccctcctg gccctccttg agtgcccacc 1666 cataaatgcg aaatgccgtc agcagacaac ggttcccacc cctggggtcc tcacaacagg 1726 ctgcatcagg gctctcgacg cttgctggct tcatccatca actgctgggt gatctcctcg 1786 aacgcatcct tgatcgcgag ttcctcgaaa tcagcggcag cttgctccca gtggatccgt 1846 tccagctccg gcagcgccgc ccacagcgcc atgcgcaagt aggttccacg tgatcgctta 1906 gtgcgctcgg cgagcgcgtc caggcgctcg atcagctccg gttccaggcg cacggagacc 1966 acggggccgc gtccggcggg gttcttctgg ttggtggcca tgagaaattt cctctcgctt 2026 cggtagttgt aaacaatgtt tacaccgtgt cggggagagg ggtttttatt tttctcccgg 2086 gcactttcga gacgggtcat gccgtaagcg aggcgcgtgg ccacaccgca ctcggcgacg 2146 caggtgtcac ttgctccccg actgtcggcc gggaaggggg cgcgcagagc gtccaggagc 2206 gccgtagagc gcctgggacg gttcgtgtgg ggacttggtc gccccacggg gctttaatcg 2266 cttaaaaacg cgcacagcgc atttcttgcc acgggctagc gcgtgaccgc tgcgcgctca 2326 cttgctcagg aagaaaatca ttcctcgcct aaagcgcttc gcgcgctcgc cctctccgag 2386 ggggaaaact aaccacacac ctcatgcact aaagtgctga tttgcaggtc agcgcgtttt 2446 agcgtgcaaa aatagtgcgg aaaacggcga aaatgggggc gcgacaatcc cctcagtggc 2506 tccccaaaat tcacctattc acatctgcta ctggctgact tctttcccga caaggggccc 2566 tgtgagggcg caggttgagc cacttttacg tcccggagat ccctttaggg cgtattcgag 2626 gtgtgctcag tgacccgctt cggcggggtg ggagtagcca aaagtccgac atttttaacg 2686 aacgttcgtt aaaatggggg catgactcag ggacctttga cctcagaaac cggcgcaatc 2746 ctgaatgatc ttggcgcagc agaccctctc gatgtggctg tccgggcacg ggagagtgcg 2806 catgttctct ctcaagtcgt ggagttttta gagcagatcg gccggtctgg ggatagcgat 2866 ttagacgcgg tgtatgagcg tgattggcag ctcgatgcag acacgttgac cttcattgcc 2926 caggcgttgg aggggttggc ggaccaggcc gaggcgaagg atgccgtgaa cgaatgacgg 2986 atatgtgtgc ccaatgcggt ggggaaatcc cgccccggcc tgacccccgc ggacgcaggg 3046 cgaagtattg ctcggatgct tgtcgggcgg cagcgagccg cgaacgcgcg cgccagcgcc 3106 acgcccagga ggtcgaagcc gcgcgtctcc aggccgcact cgatctgaaa accccgcagg 3166 agaccctggc agaggtagtc caggagcttc aggccaccac ccggattatc cgtgatcgag 3226 gggacgtgcc agcgtcgctg cgtccgctgg ttaatgctgc atccgaactg gtcaacgcag 3286 cgcaaccggt tgaggaatct aagtcattcc ccaaccggcg agtgcgtcgt gcagttaaac 3346 gaaagtttgc gataagcggg tgatgtaact gatggagatt tttacctggg ggtgtctcca 3406 gcgaggtggc caagtccgat tgtgttgagg attaccccaa acgtgcgggg attattcaaa 3466 atccactgtc caaccgcttt tccggttacc ccgcctccga tgcagcctac gagaatagag 3526 cccatgacca ttgcattgtg gctatatccc gcatttggat ccagcgccga gaaactggtg 3586 taggcaccag cagcgcagcc tgcaatgcga gcgccaatga taaccagggg gagggcgcga 3646 ggcattactc gattttcatc tgtggtctgt cgctgaatcg aagcagtgat ggcttcttca 3706 aatgcttcag ggtttgacgt ggggtccgag actgttgacg cagcttcctg cactgccttg 3766 atgaggacat cttcagggat ggaatcattg aacattcctc ccagctcaga agtggtttga 3826 acgttagccg aagggacatg cacatcgggg gaagcctggg cggctggagc aattaaagac 3886 agcgacagtg aagcaacgag agccgttaca gtggcacgag tttttaaata catgaggcga 3946 acttaacaaa ccattgatag gttgtcgtgc ggtaaagata agaaaaggat aaagatatga 4006 aaacgttatt tatgaatctc ttaggtgccg cgcttgtagg agcggtaatc atggtcttga 4066 catggttatt tattgatttt gatgcacctg gagcatggct cggattcttt attatcacca 4126 ccatcagtga ttgctgcttt agaagtcatc cacggacttt gggaaaaacg gcagggatct 4186 tccactgaca atgattgata aaacctggtt gaacggaata caaaacgcgc aaaataacca 4246 ggcagttaaa agaaaaacca gataagctgc accaatactt gaaaaatgtt gaacgccccg 4306 acagctgtaa ctgtcgaggc gtcggctaac ccccagtcat cagctgggag aaagcactca 4366 aaa 4369 10 492 PRT Corynebacterium thermoaminogenes 10 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp 50 55 60 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe 145 150 155 160 Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 Arg Leu Ser Arg Trp Pro Leu His Val Phe Asp Asp Pro Thr Ala Tyr 195 200 205 Ser Trp His Cys Gln His Asn Arg Val Asp Ile Leu Asp Glu Leu Met 210 215 220 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Lys His Ala 225 230 235 240 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 285 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln 325 330 335 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu 385 390 395 400 Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln 405 410 415 Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr 420 425 430 Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu 435 440 445 Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser 450 455 460 Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys 465 470 475 480 Cys Gln Ile Leu Arg Gly Ala His Ala Val His Arg 485 490 11 22 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 11 aaccaggggg agggcgcgag gc 22 12 26 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 12 tctcgtaggc tgcatccgag gcgggg 26 13 32 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 13 gctctagagc aaccaggggg agggcgcgag gc 32 14 36 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 14 gctctagagc tctcgtaggc tgcatcggag gcgggg 36 15 32 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 15 gctctagagc aaccaggggg agggcgcgag gc 32 16 36 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 16 gctctagagc tctcgtaggc tgcatcggag gcgggg 36 17 32 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 17 cccgttaact gcttgaaacc caggacaata ac 32 18 30 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 18 cccgttaaca tgtacttcag aaaagattag 30 19 26 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 19 gatatctacg tgccgatcaa cgtctc 26 20 25 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 20 aggccttttt ttaaggcagt tattg 25 

We claim:
 1. An isolated plasmid comprising a gene, said gene encoding a polypeptide having Rep protein activity and, said polypeptide comprising an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO:
 8. 2. The plasmid according to claim 1, wherein said polypeptide comprises an amino acid sequence that is at least 99% homologous to the amino acid sequence of SEQ ID NO:
 8. 3. The plasmid according to claim 3, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:
 8. 4. The plasmid according to claim 1, wherein said plasmid is isolated from Corynebacterium thermoaminogenes AJ12309.
 5. The plasmid according to claim 1, which has the restriction map shown in FIG.
 2. 6. The plasmid according to claim 1, wherein said gene is obtained from a plasmid having the restriction map shown in FIG.
 2. 7. The method of isolating the plasmid according to claim 1, comprising (A) culturing a Corynebacterium thermoaminogenes in a culture medium, (B) obtaining fractions by an alkali method, and (C) isolating said plasmid.
 8. The method according to claim 7, further comprising analyzing the fractions by agarose gel electrophoresis.
 9. An isolated polynucleotide comprising a nucleic acid sequence that encodes a polypeptide having Rep protein activity and, said polypeptide comprising an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO.
 8. 